Post-translational modifications can have profound effects on the activities of proteins. One type of protein modification involves the transfer of methyl groups to a specific arginine residue resulting in the formation of dimethylarginine. This reaction is catalyzed by a protein arginine methyltransferase (PRMT). PRMTs have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. Arginine dimethylation has been implicated in such cellular processes as transcription (Chen et al. 1999 Science 284:2174-7), signal transduction (Bedford et al. 2000 J Biol Chem 275:16030-6; Mowen et al. 2001 Cell 104:731-41), nuclear export (Shen et al. 1998 Genes Dev. 12:679-91), myelin integrity (Kim et al. 1997 Int J Biochem Cell Biol 29:743-51) and possibly antigenicity (Brahms et al. 2000 J Biol Chem 275:17122-9).
Methylation of arginine residues is one of many covalent modifications of eukaryotic proteins that occur concomitant with or shortly following translation. There are two classes of PRMTs that differ in the type of dimethylarginine that they produce. Type I PRMTs produce asymmetric NG, NG dimethylarginine residues, while type II PRMTs produce symmetric NG, Nxe2x80x2G dimethylarginines (FIG. 1). See also Gary et al. 1998 Prog. Nucleic Acid Res. 61:65-133).
Most substrates for type I enzymes bind nucleic acid, usually RNA. These include heterogeneous nuclear RNA binding proteins (hnRNPs), which collectively contain 65% of the nuclear asymmetric dimethylarginine, as well as fibrillarin and nucleolin (Lischwe et al. 1985 J. Biol. Chem. 260:14304-14310; Liu et al. 1995 Mol. Cell. Biol. 15:2800-2808; Najbauer et al. 1993 J. Biol. Chem. 268:10501-10509). Examples of physiological substrate of symmetric (type II) arginine methyltransferase include myelin basic protein, a major protein component of the myelin sheath, as well as the Sm proteins D1 and D3, which are components of small nuclear ribonucleoproteins.
Genes encoding rat (PRMT1), human (HRMT1L2), and yeast (RMT1) type I enzymes have been characterized (Gary et al. 1996 J. Biol. Chem. 271:12585-12594; Henry et al. 1996 Mol. Cell. Biol. 16:3668-3678; Lin et al. 1996 J. Biol. Chem. 271:15034-15044; Scott et al. 1998 Genomics 48:330-340). The mammalian genes appear to be ubiquitously expressed in all tissues (Lin et al. supra; Scott et al. supra; Tang et al. 1998 J. Biol. Chem. 273:16935-16945).
Type I enzymes have been implicated in a variety of processes, including cell growth control, signal transduction, and protein trafficking. The enzymes preferentially methylate motifs rich in arginine and glycine (RGG boxes), a common feature of the RNA binding domains of hnRNPs (Liu et al. 1995 Mol. Cell. Biol. 15:2800-2808). Arginine-methylated hnRNP A1, but not Hrp1p, has lower affinity for RNA than the native protein, suggesting a potential mechanism for modulation of protein-RNA interactions. Levels of protein methylarginine may change in response to extracellular stimuli under circumstances in which biological responses are also suppressed by methyltransferase inhibitors. These include nerve growth factor-induced neurite outgrowth in PC12 cells and mitogenic responses of lipopolysaccharide-treated B cells.
Interactions between the PRMT1 enzyme and potential signaling components have also emerged from yeast two-hybrid screens. The immediate-early gene product TIS21 (BTG2) and the leukemia-associated gene product BTG1 interact with PRMT1 and can modulate its enzymatic activity in vitro. TIS21 and BTG1 both belong to a family of mitogen-induced proteins implicated in negative regulation of the cell cycle. PRMT1 also binds to the cytoplasmic domain of the IFNAR1 chain of the alpha beta interferon receptor, while growth-inhibitory effects of interferon were suppressed by antisense oligonucleotides directed against the methyltransferase. Finally, a novel arginine methyltransferase (CARM1) associates with p160 coactivators and serves as a secondary coactivator of nuclear hormone receptors.
Other studies have identified a role for arginine methylation in protein trafficking. Shuttling of the yeast hmRNP-related proteins Np13p and Hrp1p between the nucleus and cytoplasm requires methylation by the Hmt1p methyltransferase. The human enzyme complements the shuttling defect, suggesting functional conservation between the two enzymes. Nuclear translocation of the large form of basic fibroblast growth factor may also depend on arginine methylation. In the presence of a methyltransferase inhibitor, basic fibroblast growth factor was not methylated and the protein did not localize to the nucleus.
The prevalence of NG,NG-dimethylarginine in RNA binding proteins and conservation among protein arginine N-methyltransferases underscore the potential biological importance of this posttranslational modification. However, a major issue arguing against a dynamic role for the type I enzymes in cell regulation concerns the possibility that arginine methylation is both constitutive and irreversible. While most substrates have not been characterized, some are known to exist only in a fully methylated state. Moreover, no demethylase capable of removing dimethylarginine residues has been identified and in the case of histones, turnover of dimethylarginine accompanies protein degradation.
Efforts to understand the biochemical function of mammalian arginine methyltransferases are complicated by several factors, including the existence of multiple enzymes and the fact that methyltransferase inhibitors nonspecifically target multiple processes in which S-adenosylmethionine serves as a methyl donor. In yeast, functional studies of arginine methylation have benefited greatly from genetic approaches that have led to the isolation of cells deficient in the enzyme. In principle, gene targeting strategies could be used for similar studies of the mammalian enzymes, assuming that the proteins are not required for cell viability.
It is likely that the different classes of PRMTs will regulate different cellular targets and pathways as the known substrates for class I and class II PRMTs appear to be distinct from each other. It is an object of the present invention to provide methods and systems that can detect methylarginine residues in polypeptide samples, and can differentiate between symmetric and asymmetric dimethylation.
One aspect of the present invention provides a method for identifying the structure of a dimethylarginine, e.g., to distinguish between symmetrical and asymmetrical dimethylarginine residues. In certain embodiments, the subject method includes obtaining, by mass spectroscopy, a neutral loss spectra of a peptide containing a dimethylarginine. From the neutral loss spectra, the neutral loss, if any, of monomethylamine, dimethylcarbodiimide, and/or the neutral loss of dimethylamine is determined. Neutral loss of monomethylamine and dimethylcarbodiimide indicates the presence of a symmetrically dimethylated arginine residue. On the other hand, neutral loss of dimethylamine indicates the presence of a asymmetrically dimethylated arginine residue.
Another aspect of the present invention provides a method for identifying dimethylarginine residues in a test peptide. In general, the method includes identifying the presence of dimethylarginine residues from mass spectra of a sample peptide obtained under conditions in which the spectra reveal mass modification by methylation, if any, of arginine residue in the sample peptide. For dimethylarginine residue which are identified, the method also ascertains the nature of the methylation by determining if a neutral loss spectra of the sample peptide shows one or both of neutral loss of monomethylamine, dimethylcarbodiimide, and/or neutral loss of dimethylamine. Neutral loss of monomethylamine and of dimethylcarbodiimide indicates the presence of a symmetrically dimethylated arginine residue whereas neutral loss of dimethylamine indicates the presence of a asymmetrically dimethylated arginine residue.
Still another aspect of the invention provides a method for identifying substrates of a protein arginine methyltransferase. According to the subject embodiment, mass spectra are obtained for one or more sample polypeptides which have been exposed to a protein arginine methyltransferase (PRMT) under conditions wherein methylation, if any, of arginine residues in a substrate protein of the PRMT can occur. The presence of dimethylarginine residues in the sample polypeptide can be identified by the presence of mass modified arginine (relative to unmodified arginine) in the mass spectra of the sample protein. To ascertain the nature of the methylation of a dimethylarginine residue, further determines if a neutral loss spectra of the sample peptide shows one or both of neutral loss of monomethylamine, dimethylcarbodiimide, and/or neutral loss of dimethylamine. Neutral loss of monomethylamine and of dimethylcarbodiimide indicates the presence of a symmetrically dimethylated arginine residue whereas neutral loss of dimethylamine indicates the presence of a asymmetrically dimethylated arginine residue. In certain preferred embodiments, the sequence of at least that portion of a polypeptide including a dimethylarginine residue is determined, e.g., based on the mass spectra of the polypeptide. The subject method can be carried out for a library of sample polypeptides.
Thus, the invention provides A method for identifying substrates of a protein arginine methyltransferase comprising: (i) obtaining mass spectra for one or more sample polypeptides which have been exposed to a protein arginine methyltransferase (PRMT) under conditions wherein methylation, if any, of arginine residues in a substrate protein of the PRMT can occur; (ii) identifying the presence of dimethylarginine residues in the sample polypeptide by the presence of mass modified arginine in the mass spectra; and (iii) ascertaining the nature of the methylation of a dimethylarginine residue identifying in step (i) by determining if a neutral loss spectra of the sample peptide shows one or both of neutral loss of monomethylamine (MMA), dimethylcarbodiimide (DMC), and/or neutral loss of dimethylamine (DMA), wherein neutral loss of monomethylamine (MMA) and of dimethylcarbodiimide (DMC) indicates the presence of a symmetrically dimethylated arginine residue whereas neutral loss of dimethylamine (DMA) indicates the presence of a asymmetrically dimethylated arginine residue.
In certain embodiments, the method further comprising determining the substrate specificity of a protein arginine methyltransferase by determining the sequence of at least that portion of a polypeptide including a dimethylarginine residue.
In certain embodiments, said one or more sample polypeptides constitute a library of sample polypeptides.
In certain embodiments of the above methods, the neutral loss spectra is generated from the mass spectra.
In certain embodiments of the above methods, the spectra are obtained using a mass spectrometer in which ionization of the sample protein is accomplished matrix-assisted laser desorption (MALDI) ionization, electrospray (ESI), or electron impact (EI). In certain preferred embodiments, the spectra are obtained using ESI-MS/MS.
Another aspect of the invention provides mass spectrometry system including an automated system for identifying dimethylarginine residues in a test peptide, which automated system comprises: (i) a methylation detection system that (a) analyzes one or more mass spectra obtained under conditions in which the spectra reveal mass modification by methylation, if any, of arginine residue in a sample peptide, and (b) identifies the presence of dimethylarginine residues from characteristic mass differences between arginine and dimethylarginine in the mass spectra; (ii) a neutral loss detection system for determining the nature of the methylation of a dimethylarginine residue identified by the methylation detection system, which neutral loss detection system determines if a neutral loss spectra of the sample peptide shows one or both of neutral loss of monomethylamine (MMA), dimethylcarbodiimide (DMC), and/or neutral loss of dimethylamine (DMA), wherein neutral loss of monomethylamine (MMA) and of dimethylcarbodiimide (DMC) indicates the presence of a symmetrically dimethylated arginine residue, whereas neutral loss of dimethylamine (DMA) indicates the presence of a asymmetrically dimethylated arginine residue.
Another aspect of the invention provides a method of conducting a drug discovery business. The method utilizes any of the above methods to determine the identity of a PRMT and substrate thereof. Agents are identified by their ability to alter the level of methylation of the substrate. Therapeutic profiling of identified agents, or further analogs thereof, for efficacy and toxicity in animals are accomplished, and pharmaceutical preparations are formulated including one or more agents identified as having an acceptable therapeutic profile. The subject method can include an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.
Thus the invention provides A method of conducting a drug discovery business, comprising: (i) by the method of any of claims 1-8, determining the identity of a PRMT and substrate thereof; (ii) identifying agents by their ability to alter the level of methylation of the substrate; (iii) conducting therapeutic profiling of agents identified in step (ii), or further analogs thereof, for efficacy and toxicity in animals; and (iv) formulating a pharmaceutical preparation including one or more agents identified in step (iii) as having an acceptable therapeutic profile.
In one embodiment, the method further includes an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.
Yet another aspect of the present invention provides a method of conducting a proteomics business. The method utilizes any of the above methods to determine the identity of a PRMT and substrate thereof. Rights for further drug development of agents that alter the level of methylation of the substrate are licensed to a third party.
Another aspect of the present invention relates to a protein sequence or coding sequence database comprising annotation information representative of the methylation status (see below) of arginine residues or arginine-containing polypeptides.
Still another aspect of the present invention provides a mass spectrometry system including an automated system for identifying dimethylarginine residues in a test peptide. The automated system generally includes: (i) a methylation detection system that (a) analyzes one or more mass spectra obtained under conditions in which the spectra reveal mass modification by methylation, if any, of arginine residue in a sample peptide, and (b) identifies the presence of dimethylarginine residues from characteristic mass differences between arginine and dimethylarginine in the mass spectra; (ii) a neutral loss detection system for determining the nature of the methylation of a dimethylarginine residue identified by the methylation detection system, which neutral loss detection system determines if a neutral loss spectra of the sample peptide shows one or both of neutral loss of monomethylamine, dimethylcarbodiimide, and/or neutral loss of dimethylamine.
In one embodiment, the methylation detection system comprises a commercially available mass spectrometer possessing the ability to detect post translational modification, such as methylation. In another embodiment, the neutral loss detection system is part of a commercial mass spectrometer (such as a Thermo Finnigan mass spectrometer).
In preferred embodiments, the methylation detection and neutral loss detection systems are software components running on a computer which acquires mass spectra from a spectrometer.